The present invention relates generally to the field of drug delivery systems. More particularly, the present invention relates to a method for microencapsulating drugs using biodegradable nonantigenic materials and also to microencapsulated compositions that are targeted either to macrophages, other phagocytic cells of the immune system, or a diseased organ, which phagocytize the microspheres and digest the coating, releasing the intact drug or active fragment thereof intracellularly or at the site of attachment Such compositions are useful in treating and preventing disease.
Drug delivery technology can bestow new leases on the lives of seemingly ineffective or inefficient drugs by targeting them specifically to sites of action. In this manner, unwanted systemic side effects are obviated and dose requirements are substantially reduced. Macrophage mediated delivery of drugs has been suggested as an alternative for treatment of several types of diseases. Modern immunology has acknowledged the importance of the monocyte and its mature counterpart, the macrophage as the prime antigen presenting cell in immune interactions. As an extension of this function it can be theorized that macrophages may be utilized to present immunoactive drugs to relevant components of the immune system such as lymphocytes in an effort to modulate their function.
Systemic elimination systems developed by the body to attack and eliminate foreign material include macrophages and Kupffer cells. For the purposes of the present invention the term macrophage includes Kupffer cells, when appropriate. Macrophages are cells widely distributed in many tissues of the body including lympho-hemopoietic organs, skin, gut, other portals of entry and the nervous system. They are found in direct contact with the blood (monocytes, sinus-lining Kupffer cells) or extravascular space, and undergo complex migrations as they enter tissues after production mainly within the bone marrow of the adult. As mobile and long-lived cells the mononuclear phagocytes play central roles as effector cells in inflammatory reactions and cell mediated immune responses. Monocytes produced in the bone marrow are released into the blood stream where they circulate with an estimated half life of 8-9 hours. Most experimental evidence indicates that the monocytes migrate into tissue to replace senescent tissue macrophages, and differentiate into cells of varied morphological and functional characteristics. Although definitive information on density and tissue distribution of these cells is still not available it is clear that the tissue content of mononuclear phagocytes, often associated with the vasculature greatly exceeds the bone marrow replicative pool. These cells, in a real sense are the first line of defense, followed by secondary waves of granulocytes, lymphocytes and monocytes from the circulation.
The term xe2x80x9cactivated macrophagexe2x80x9d refers to cells with increased phagocytic activity, increased content of acid hydrolases, more active metabolism, and, more importantly, an increased microbicidal capacity. Macrophages become activated following specific immunity involving the T lymphocyte. The T lymphocyte represents the specific branch of the cellular response which, in some way or another (most likely via secretion of soluble products), activates macrophages. The activated macrophages display considerable enhancement in their capacity to curb bacterial growth. Although macrophage activation clearly follows immune activation of the T cells, it can also result from direct interaction of certain bacterial products or other chemicals with the macrophage. Unstimulated macrophages or non-activated macrophages generate low and variable cytokine activity. However, as with enzyme secretion, the secretion of these lymphocyte-stimulatory activities can be modulated. Two conditions produce an increase in activity: one is a phagocytic challenge, the second is exposure to activated T lymphocytes. Exposure of macrophages to bacteria, antigen-antibody complexes, latex beads, or endotoxin results in a marked increase (up to 10 times) in the activity tested, both in mitogenic response of the thymocytes and in antibody formation. This increase is seen for 1 to 2 days, and then decays away. The presence of activated T cells, which by themselves were not responsible for the activities, markedly enhances their production. The highest mitogenic activity found in cell culture results from the addition of a small number of the activated T cells to the macrophage culture.
The term phagocytosis is used to describe the internalization of large particles, such as those visible by light microscope, mostly viruses and bacteria. Uptake occurs by close apposition of a segment of plasma membrane to the particle""s surface, excluding most if not all of the surrounding fluid. The phenomenon of phagocytosis seen in living cells in tissue culture or in-vivo has been well described. Phagocytosis is a process that occurs in three stages: attachment, ingestion, and digestion of the particles. The activated macrophage possessing a ruffled surface at the leading edge pushes out processes towards a particulate substance and rapidly flows around it. The entire process may only take a few minutes. Once ingested, the material may be totally digested, it may persist in the form of an indigestible residue, or it may actually fill up the whole cell, and if toxic may kill the cell. When the particle is too large for one cell to ingest, several cells flow around it and form a capsule. After ingestion, the vesicle which forms around the phagocytized particle, the phagosome fuses with one or more iysosomes to form a secondary lysosome or phagolysosome. The lysosome is a membranous bag of hydrolytic enzymes to be used for the controlled intracellular digestion of ingested materials. The hydrolytic enzymes contained in the lysosome are thus discharged into the enlarged vacuole to degrade the contents.
The deleterious effects of immune modulated diseases that manifest themselves due to improper recognition of xe2x80x9cselfxe2x80x9d from xe2x80x9cnon-selfxe2x80x9d may be effectively reduced using this mode of drug delivery. Rheumatoid arthritis (RA) is one such disease affecting a large percent of the geriatric population. And while macrophages may not be a primary target for the virus causing acquired immune deficiency syndrome (AIDS), they have been implicated as carriers of the virus.
Cytokines are polypeptide hormones which have a variety of physiologic activities intended to xe2x80x9cup-regulatexe2x80x9d and/or xe2x80x9cdown-regulatexe2x80x9d the immune system.
The cytokine cascade, although not fully elucidated, involves the release of a number of molecules, including tumor necrosis factor alpha (TNFa), interleukin-1 beta (IL1xcex2), interleukin-2 (IL2), interleukin-6 (IL6), interleukin-8 (IL8), colony stimulating factors (CSF), interferons (IFN), interleukin-1 receptor antagonist (IL1-ra) as well as other interleukins whose function is still not completely understood from monocytes, macrophages, lymphocytes, and other tissues throughout the body. These cytokines act both locally and systemically to recruit other white blood cells to the site of infection, activate macrophages, increase antibody production, produce fever, hyperlipidemia, hyperglycemia, and directly activate natural killer and lymphokine activated killer lymphocytes thereby destroying tumor cells. In cancer patients, the concentration of cytokines such as tumor necrosis factor alpha (TNFa) and interleukin-1 beta (IL1-xcex2) have been observed to be decreased during active tumor growth. Administration of macrophage/immune activators such as microencapsulated macrophage colony stimulating factor have been shown to reduce cancer mortality and lead to increases in local TNFa and IL1xcex2 concentration. Alternatively, administration of microencapsulated TNFa and IL1xcex2 may activate cellular immunity and increase tumoricidal activity of T-lymphocytes through upregulation of macrophage induced T-cell clonal expansion.
Although this response is usually beneficial, during overwhelming sepsis or other immune response challenges a hyper-responsiveness of cytokines has been shown to produce lethal effects. Massive vascular permeability leads to cardiovascular collapse and pulmonary edema. Coagulation defects may also complicate the clinical condition adding to increased mortality. It has been demonstrated in several animal models that the pre-treatment and continued intravenous administration of monoclonal antibodies (MAB""s) or polyclonal antibodies (PAB""s) to TNFa or IL1-ra can attenuate the clinical syndrome of sepsis and prevent mortality from lethal injections of live bacteria.
The use of intravenously administered MAB""s to cytokines have been reported in the scientific literature for the prevention of mortality from experimental septic or endotoxemic shock since 1987. Typical experiments have used monoclonal antibodies, such as those to TNFa, administered directly to a subject concomitant with a dose of bacteria or other source of immunogenic challenge.
There are several potential problems with the direct administration of high, frequent doses of MAB""s for systemic inhibition of cytokines. Although animal models provide an in vivo system to test a septic/endotoxemic model, sepsis in humans is sudden in onset and originates from a localized infection site rather than by an intravenous challenge. The current method of administration requires an antigen-antibody reaction with the cytokine in systemic circulation (i.e., after synthesis and release by the macrophage). Since intravenous MAB""s can inactivate systemic cytokines, there is a potential for intravenous MAB""s to inhibit the autocrine up-regulation of further cytokine synthesis and release by activated macrophages and lymphocytes. However, it is doubtful that unencapsulated MAB""s can gain sufficient access to the macrophage localized at the site of infection to prevent release of the cytokines. Furthermore, MAB""s administered systemically typically are metabolized before they reach the desired site.
Prior art efforts to target delivery of MAB""s for reduction of inflammatory response have yet to effectively deliver the molecules into the macrophage to the area of localized infection or immune response, where cytokine production is initiated. Other immunologic pathogenic responses such as lupus, organ transplant rejection, and glomerulonephropathies have been shown to have localized cytokine production as the major cause of tissue inflammation and ultimate destruction of the organ involved.
Since reduction in cytokine response has been shown to lead to a decrease in the severity of several diseases, it would be desirable to have a targeted drug delivery system that would act intracellularly at a targeted site, rather than systemically. Such a system would utilize an encapsulating material that is recognizable by the uptake cell and be transported intracellularly where it would release the encapsulated drug into the cytoplasm.
It is therefore a principal object of the present invention to provide a delivery system for drugs and other molecules for cellular uptake (e.g., macrophages and Kupffer cells).
It is a further object of the present invention to provide a delivery system using albumin as an encapsulating material to coat a drug such that when administered in vivo the microsphere will be ingested by a macrophage, resulting in the digestion of the albumin and the release of the drug or an active form or fragment of the drug intracellularly.
It is another object of the present invention to provide a method for treating an immune modulated disease comprising administering to a subject a preparation containing pharmaceutically acceptable carrier and a biodegradable microsphere containing a neutralizing antibody to a cytokine.
As an example of the use of a drug as an immunosuppressant, Cyclosporin A (CsA) (Sandimmune, Sandoz) has gained wide acceptance by most transplant physicians as the immunosuppressant of choice for preventing rejection of solid organ grafts and graft-versus-host disease. The drug has a specific effect on T-lymphocytes in which it seems to prevent the transcription of genes for several lymphokines. The reduction in IL-2 prevents the clonal expansion of T-lymphocytes and their differentiation into effector T-cells. The reduction in IFN-tau interrupts the feedback mechanism between T-cells and macrophages and the aberrant expression of major histocompatibility complex (MHC) class II molecules. Through these mechanisms CsA exerts an immunosuppressive and anti-inflammatory effect
Considerable evidence has accumulated to suggest that rheumatoid arthritis (RA) is an auto-immune disease. Activated T-lymphocytes interrelate with macrophages, other inflammatory cells and effector cells in joint tissue, leading to symptoms of inflammation accompanied by joint destruction. Immunosuppressive treatment is already well established in this disease and several trials have already taken place using CsA. A review of studies concludes that CsA is efficacious in controlling inflammatory and functional symptoms, although this improvement is not generally accompanied by reductions in erythrocyte sedimentation rate (ESR) and rheumatoid factor. The frequency of adverse events is comparable to that of other treatments but nephropathy remains the principal factor limiting the use of CsA. Recent evidence suggests that with a strict dosage strategy and good monitoring this problem is controllable and reversible.
Studies have also shown that CsA is capable of inhibiting both adjuvant arthritis and collagen arthritis in rats when administered at the time of disease induction. The effects, however, on established arthritis in these animals appear to be different depending on the animal model. Toxicity is another major concern associated with the use of CsA. Nephrotoxicity, hepatotoxicity, hirsutism, neurotoxicity, hypertension and altered coagulability have all been reported with CsA. In RA patients treated with CsA, those adverse effects that are the most prevalent include nephrotoxicity, hypertension, gastrointestinal intolerance, hypertrichosis and tremors due to high systemic levels of CsA. Recent reports indicate that the clearance of CsA is decreased in animals models of arthritis and diabetes. Therefore, when treating RA patients with CsA, the dose and blood levels of this drug should be carefully monitored because of drug toxicity, and because of the effect of the disease state on the disposition of the drug. A targeted microsphere system employing subtherapeutic doses of CsA would be desirable in order to overcome the limitations of systemic, nonspecific delivery of CsA. Other immunologically mediated diseases such as glomerulonephritis, organ transplant rejection, and lupus would likely benefit from a similar application of microencapsulated immunosuppressive agents.
Prior art encapsulation techniques have been directed at sustained release of an encapsulated drug using a selectively permeable coating or membrane through which the drug dissolves or otherwise passes. Numerous patents and articles describe the encapsulation of a variety of molecules, including drugs, vitamins, hormones, steroids, viruses, and other compositions by an equally numerous variety of coatings or membrane materials. For instance, U.S. Pat. No. 5,017,379 describes antifibrin antibodies encapsulated in a biodegradable microcontainer which are released at the site of a targeted blood clot in an artery or vein. U.S. Pat. No. 4,925,661 discloses a method of delivering cytotoxic reagents, such as the A fragment of the diphtheria toxin, to cells by encapsulating the toxin in an immunoliposome composed from phosphatidylethanolamine and oleic acid in a molar ratio of 8:2 and a fatty acid derived antibody.
The present invention relates generally to an improved drug delivery system for modulating immune response by modifying cytokine response in macrophages. More particularly, a composition is disclosed for use in a drug delivery system comprising a drug microencapsulated with a biodegradable nonantigenic substance that can be recognized, phagocytosed and uncoupled by macrophages. In a preferred embodiment, the present invention provides a anticytokine neutralizing antibody (NA) that is encapsulated with albumin to form a microsphere. The microsphere is formulated into an injectable or otherwise administrable preparation that can be administered in vivo. Once administered the microspheres are recognized and attacked by macrophages and ingested by phagocytosis. Once inside the macrophage enzymes digest the albumin, releasing the NA or an active form or fragment of the NA into the cell interior. NA may bind to cytokine receptors or translated cytokines inhibiting further activity. Microencapsulation techniques presented herein may also be used to target the macrophage with other agents such as proteases or anti-retroviral agents used to treat other illnesses which as systemic in nature. Diseases such as septic shock, which result from a massive immune reaction from invasion by bacteria, fungi, or bacteria-produced endotoxin or exotoxins, can be effectively treated by this composition.
A potential mechanism of action of microencapsulated NA on endotoxin or bacteria stimulated cytokine release involves the following steps and is depicted in FIG. 1. Step 1: Endotoxin/bacteria are either phagocytized or bind to the macrophage. Step 2: The organism/product transduces a signal to the nucleus stimulating transcription and translation of TNFa, IL-1xcex2, IL-6 and IL-8. Step 3: TNFa is synthesized and released into the cell where it can either be secreted or re-stimulate the nucleus to produce additional cytokines (autocrine). Step 4: Phagocytized microcapsules of NA are uncoupled by lysosomal enzymes and the active F(ab)2 fragments bind to the intracellular cytokine inhibiting both autocrine up-regulation and release of the active cytokine. Binding of TNF-NA or IL1-NA may reduce related cytokines through an inhibition of nuclear stimulated cytokine synthesis (see Step 5) or by binding prevent released cytokine from binding to extracellular receptors (see Step 7), which has been shown to occur in vivo. Step 5: TNFa, IL-1xcex2, and IL-6 have been shown in vivo to stimulate synthesis and release of each other probably by nuclear up-regulation of cytokine synthesis. Step 6: Released, unbound cytokines can stimulate effector organs as well as bind to lymphocytes and other white cells stimulating further production of cytokines. Step 7: Cytokine binding of receptors of macrophages and other white cells can lead to an increase in cellular synthesis and release of cytokines. Step 8: Membrane bound cytokines stimulate nuclear synthesis of all the cytokines in the cascade. Summary: Microencapsulated anticytokine antibodies may exhibit their effect on cytokine release by acting at Steps 3 and 5 and therefore blunt further release at Steps 6, 7, and 8.
The present invention also may be particularly useful in treating rheumatoid arthritis, AIDS, uveitis, psoriasis, arthritis, diabetes, asthma, organ transplant rejection, lupus, tumor regression, viral infections, renal disease such as glomerulonephropathies, presentation of vaccines and adjuvants, myasthenia gravis and other immune modulated conditions.
Albumin or globulin is the preferred material used to encapsulate water soluble molecules, such as anticytokine NA, peptides, insulin, proteins, drugs and the like. In an alternative embodiment, poly DL(lactic co-glycolic acid) (PLGA), a linear polyester that breaks down to D-lactic acid, L-lactic acid and glycolic acid, is used to encapsulate water insoluble molecules, such as vitamins and many drugs.
In another aspect of the present invention, a method is presented for preparing the encapsulated drug comprising dissolving albumin in water or phosphate buffered saline (PBS); separately solubilizing NA in PBS; mixing the two solutions together; cooling the mixture; adding the mixture to an amount of olive oil previously cooled, emulsifying the mixture to form microspheres; crosslinking the microspheres by adding a solution of glutaraldehyde and stirring while maintaining the cool temperature of the mixture; washing the microspheres; sizing the microspheres; and drying the microspheres.